scholarly journals Two structurally and kinetically distinct forms of Wolinella succinogenes nitrite reductase

1990 ◽  
Vol 271 (1) ◽  
pp. 259-264 ◽  
Author(s):  
R S Blackmore ◽  
T Brittain ◽  
P M A Gadsby ◽  
C Greenwood ◽  
A J Thomson
Keyword(s):  
Electron Transfer ◽  
High Spin ◽  
Spin State ◽  
Nitrite Reductase ◽  
Exchange Coupling ◽  
Methyl Viologen ◽  
Rapid Rate ◽  
Wolinella Succinogenes

It is shown that the oxidized form of the hexa-haem nitrite reductase of Wolinella succinogenes exists in two structurally and functionally distinct forms, termed ‘resting’ and ‘redox-cycled’. The nitrite reductase as initially isolated, termed ‘resting’, has five low-spin ferrihaem groups and one high-spin ferrihaem group. The reduction of these haem groups by Na2S2O4 occurs in two kinetically and spectrally distinct phases. In the slower phase the haem groups are reduced by dithionite with a limiting rate of 4 s-1. If the enzyme is re-oxidized after reduction with dithionite or with methyl viologen, the resulting ferric form, termed ‘redox-cycled’, possesses only low-spin haem centres and a rate of reduction in the slower phase that is no longer limited. In the resting form of the enzyme the high-spin ferrihaem group is weakly exchange-coupled to a low-spin haem group. It is proposed that in the redox-cycled form the exchange coupling occurs between two low-spin ferric haem groups. This change in spin state allows a more rapid rate of electron transfer to the coupled pair.

scholarly journals Storage, transport, release: heme versatility in nitrite reductase electron transfer studied by molecular dynamics simulations

2015 ◽  
Vol 17 (6) ◽  
pp. 4483-4491 ◽  
Author(s):  
Anna Bauß ◽  
Thorsten Koslowski
Keyword(s):  
Molecular Dynamics ◽  
Electron Transfer ◽  
Molecular Dynamics Simulations ◽  
Campylobacter Jejuni ◽  
Nitrite Reductase ◽  
Thermodynamic Integration ◽  
Wolinella Succinogenes ◽  
Dynamics Simulations ◽  
Kinetics Of

Using molecular dynamics simulations of the thermodynamic integration type, we study the energetics and kinetics of electron transfer through the nitrite reductase enzyme of Sulfurospirillum deleyianum, Wolinella succinogenes and Campylobacter jejuni.

scholarly journals The purification and some equilibrium properties of the nitrite reductase of the bacterium Wolinella succinogenes

1986 ◽  
Vol 233 (2) ◽  
pp. 547-552 ◽  
Author(s):  
R Blackmore ◽  
A M Roberton ◽  
T Brittain
Keyword(s):  
High Spin ◽  
Nitrite Reductase ◽  
Reductase Activity ◽  
Spin Transition ◽  
Spectral Characteristics ◽  
High Yield ◽  
Wolinella Succinogenes ◽  
Hydrophobic Binding ◽  
Nitrite Reductase Activity ◽  
Oxygen Reductase

The bacterium Wolinella succinogenes produces a nitrite reductase enzyme that can be purified to homogeneity in high yield by a combination of detergent extraction, hydroxyapatite chromatography and Mr fractionation. Nitrite reductase activity is found to be present in both a high- and a low-Mr fraction. The high-Mr fraction has been shown to consist of the low-Mr nitrite reductase enzyme associated with a hydrophobic ‘binding protein’. The amino acid composition for both proteins is reported. The nitrite reductase enzyme shows spectral characteristics indicative of the presence of c-type haem groups. Measurements at 610 nm indicate the presence of some high-spin haem groups at neutral pH. This haem subgroup undergoes a pH-linked high-spin - low-spin transition at alkaline pH. Approximately two of the six haem groups present within the enzyme bind CO with low affinity (KD = 0.4 mM). The enzyme also shows a range of redox activities with various inorganic reagents. The enzyme has been shown to exhibit dithionite reductase, oxygen reductase and CO2 reductase activities.

scholarly journals An analysis of the reaction kinetics of the hexahaem nitrite reductase of the anaerobic rumen bacterium Wolinella succinogenes

1990 ◽  
Vol 271 (2) ◽  
pp. 457-461 ◽  
Author(s):  
R S Blackmore ◽  
T Brittain ◽  
C Greenwood
Keyword(s):  
Electron Transfer ◽  
Reaction Kinetics ◽  
Nitrite Reductase ◽  
Rate Constants ◽  
Rumen Bacterium ◽  
Square Root ◽  
Wolinella Succinogenes ◽  
Single Turnover ◽  
Internal Electron ◽  
Kinetics Of

The reduction kinetics of both the resting and redox-cycled forms of the nitrite reductase from the anaerobic rumen bacterium Wolinella succinogenes were studied by stopped-flow reaction techniques. Single-turnover reduction of the enzyme by dithionite occurs in two kinetic phases for both forms of the enzyme. When the resting form of the enzyme is subjected to a single-turnover reduction by dithionite, the slower of the two kinetic phases exhibits a hyperbolic dependence of the rate constant on the square root of the reductant concentration, the limiting value of which (approximately 4 s-1) is assigned to a slow internal electron-transfer process. In contrast, when the redox-cycled form of the enzyme is reduced by dithionite in a single-turnover experiment, both kinetic phases exhibit linear dependences of the rate on the square root of dithionite concentration, with associated rate constants of 150 M-1/2.s-1 and 6 M-1/2.s-1. Computer simulations of both the reduction processes shows that no unique set of rate constants can account for the kinetics of both forms, although the kinetics of the redox-cycled species is consistent with a much enhanced rate of internal electron transfer. Under turnover conditions the time course for reduction of the enzyme, in the presence of millimolar levels of nitrite and 100 mM-dithionite, is extremely complex. A working model for the mechanism of the turnover activity of the enzyme is proposed which very closely describes the reaction kinetics over a wide range of substrate concentrations, as shown by computer simulation. The similarity in the action of the nitrite reductase enzyme and mammalian cytochrome c oxidase is commented upon.

Catalytic and spectroscopic analysis of blue copper-containing nitrite reductase mutants altered in the environment of the type 2 copper centre: implications for substrate interaction

2001 ◽  
Vol 353 (2) ◽  
pp. 259-266 ◽  
Author(s):  
Miguel PRUDÊNCIO ◽  
Robert R. EADY ◽  
Gary SAWERS
Keyword(s):  
Electron Transfer ◽  
Enzyme Activity ◽  
Nitrite Reductase ◽  
Methyl Viologen ◽  
Spectroscopic Properties ◽  
Sodium Dithionite ◽  
Epr Spectrum ◽  
Characteristic Type

The blue dissimilatory nitrite reductase (NiR) from Alcaligenes xylosoxidans is a trimer containing two types of Cu centre, three type 1 electron transfer centres and three type 2 centres. The latter have been implicated in the binding and reduction of nitrite. The Cu ion of the type 2 centre of the oxidized enzyme is ligated by three His residues, and additionally has a co-ordinated water molecule that is also hydrogen-bonded to the carboxyl of Asp92 [Dodd, Van Beeumen, Eady and Hasnain (1998), J. Mol. Biol. 282, 369Ő382]. Two mutations of this residue have been made, one to a glutamic acid residue and a second to an asparagine residue; the effects of both mutations on the spectroscopic and catalytic properties of the enzyme have been analysed. EPR spectroscopy revealed that both mutants retained intact type 1 Cu centres with g‖ = 2.12 (A‖ = 0mT) and g⊥ = 2.30 (A⊥ = 6.4mT), which was consistent with their blue colour, but differed in their activities and in the spectroscopic properties of the type 2 centres. The D92E mutant had an altered geometry of its type 2 centre such that nitrite was no longer capable of binding to elicit changes in the EPR parameters of this centre. Accordingly, this mutation resulted in a form of NiR that had very low enzyme activity with the artificial electron donors reduced Methyl Viologen and sodium dithionite. As isolated, the EPR spectrum of the Asp92 → Asn (D92N) mutant showed no characteristic type 2hyperfine lines. However, oxidation with iridium hexachloride partly restored a type 2 EPR signal, suggesting that type 2 copper is present in the enzyme but in a reduced, EPR-silent form. Like the Asp92 → Glu mutant, D92N had very low enzyme activities with either Methyl Viologen or dithionite. Remarkably, when the physiological electron donor reduced azurin I was used, both mutant proteins exhibited restoration of enzyme activity. The degree of restoration differed for the two mutants, with the D92N derivative exhibiting approx. 60% of the activity seen for the wild-type NiR. These findings suggest that on formation of an electron transfer complex with azurin, a conformational change in NiR occurs that returns the catalytic Cu centre to a functionally active state capable of binding and reducing nitrite.

Theoretical study on the reduction reactions from solid char(N): The effect of the nearby group and the high-spin state

Energy ◽  
2019 ◽  
Vol 189 ◽  
pp. 116286 ◽  
Author(s):  
Hai Zhang ◽  
Lei Luo ◽  
Jiaxun Liu ◽  
Anyao Jiao ◽  
Jianguo Liu ◽  
...  
Keyword(s):  
High Spin ◽  
Spin State ◽  
Theoretical Study ◽  
High Spin State ◽  
Reduction Reactions

scholarly journals Supercooled high spin state in metallo-supramolecular assemblies

2007 ◽  
Vol 63 (a1) ◽  
pp. s202-s202
Author(s):  
U. Pietsch ◽  
M. Lommel ◽  
Y. Bodethin ◽  
D. Kurth ◽  
G. Schwarzl ◽  
...  
Keyword(s):  
High Spin ◽  
Spin State ◽  
High Spin State ◽  
Supramolecular Assemblies
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